The goals of this Phase II research proposal focus on the further development of a topical microbicide for vaginal and rectal use from a small group of highly potent pyrimidinedione molecules identified in our Phase I research. Topical microbicides represent an important strategy for preventing the sexual transmission of HIV, the predominant mode by which HIV is transmitted worldwide, especially in the developing world. The specific rationale for the development of the pyrimidinedione series of molecules includes the observation that the compounds exhibit subnanomolar activity against clinical strains of HIV-1 and HIV-2, including subtypes found throughout the world, the compounds inhibit HIV infection and replication through two distinct mechanisms of action, including inhibition of reverse transcription and viral entry, and they display a significantly high level of activity in cervical explant models of sexual transmission. The specific aims of the proposal are constructed to identify our primary lead pyrimidinedione microbicide for clinical development from among a series of highly potent molecules which were selected based on the results of our Phase I microbicide testing. We propose to utilize this agent, either alone or in combination with another microbicide, for advancement to IND submission and clinical trials. The selection criteria used to identify the superior microbicide candidate will be based on an algorithm using established and novel in vitro assays developed to examine compound efficacy, toxicity, stability, and formulation in a microbicide-like environment. These assays will examine the efficacy of the pyrimidinediones alone and in combination with each other or other microbicide compounds against multiple clinical strains of HIV-1 and HIV-2 in the presence of vaginal and seminal simulates in primary and established cell lines hypothesized to represent models of the sexual transmission of HIV. In addition, the efficacy of the pyrimidinediones against other STIs and their toxicity to Lactobacilli and normal human vaginal and cervical cells will also be examined in an environment that mimics that of the vagina at the time of intercourse and HIV transmission. Following in vitro analysis, a lead compound will be advanced to toxicology evaluations in well established animal model systems. These animal models will utilize rabbits and rodents to examine the contact hypersensitivity and adsorption potential of the lead compounds alone and in combination. Additional studies will also be performed using the cervical explant model to confirm the efficacy and lack of toxicity of the candidate microbicide in its formulated state. These assays are designed to address the FDA's points to consider in the preclinical development of anti-infective agents, and are specifically tailored to address the requirements of an effective topical microbicide as defined by the International Working Group on Microbicides. The results of this research will be used as a basis for Phase III preclinical research and the submission of an Investigatory New Drug (IND) application to the FDA for the use of pyrimidinediones as single or combination topical microbicides. In the absence of an effective vaccine to prevent HIV infection, the development of a low cost and effective barrier to the sexual transmission of HIV is one of medicine's most pressing issues in light of the high rates of virus transmission occur in the developing world, especially in women in social settings where the negotiation of condom use is problematic. It has been estimated that a microbicide product that is only 60% effective could prevent millions of new infections by HIV each year. The pyrmidinediones have characteristics that make them superior microbicide candidates, including the fact that they are highly potent inhibitors of HIV infection, inhibit both virus entry and reverse transcription, are highly active in cervical explant models of sexual virus transmission, and they can be easily synthesized at low cost. [unreadable] [unreadable] [unreadable]